Reconstitution of normal CD4 Treg.after allogeneic HSCT provides a unique opportunity to examine and define critical elements that modulate human Treg proliferation, function and survival in vivo. The generation and maintenance of Treg function in vivo is known to be a dynamic process that is subject to complex homeostatic signals. In the context of allogeneic HSCT, deficiencies of Treg can lead to enhancement of graft versus leukemia (GVL) as well as auto-immunity and allo-immunity. Conversely, excessive Treg function can suppress GVL, increase relapse and increase susceptibility to infections. We have previously demonstrated that patients with chronic GVHD had significantly reduced frequency of Treg, Impaired Treg reconstitution in these patients was linked to specific abnormalities of Treg homeostasis in the first year post transplant, including decreased thymic generation of naive Treg, increased proliferation and increased susceptibility to apoptosis. We also demonstrated that expansion of CD4 Treg in vivo could be achieved by administration of IL-2 and completed a clinical trial of daily low-dose IL-2 in patients with refractory cGVHD. Our results suggest that low-dose lL-2 is safe in patients with active cGVHD and results in the selective expansion of CD4 Treg in vivo. Expanded Treg express FoxP3 and retain functional suppressive activity. The majority of patients treated with IL-2 noted improvement or stabilization of cGVHD. In the next 5 years. Project 3 will continue to focus on the reconstitution of Treg after allogeneic HSCT with the major goal of defining critical mechanisms that modulate Treg homeostasis in vivo. In conjunction with clinical trials designed to modulate Treg number and function after HSCT (Project 1), detailed analysis of the immunologic effects of these manipulations will lead to a better understanding of the mechanisms that control Treg homeostasis. These studies will inform the design of further clinical trials to modulate Treg function in vivo in the context of allogeneic HSCT with the goal of developing novel strategies for selectively enhancing tumor immunity, suppressing allo-immunity and improving patient outcomes. These experiments will be carried out in 4 Specific Aims: 1) To define abnormalities of Treg homeostasis that contribute to loss of tolerance and development of chronic GVHD after allogeneic HOT. 2) To identify cellular mechanisms andVsignaling pathways that modulate Treg generation, proliferation and survival in vivo. 3) To define the effects of lL-2 therapy and donor Treg infusion on Treg homeostasis after allogeneic HOT. 4) To examine effects of tumor cell vaccination and other post-transplant immunologic interventions on Treg in vivo. RELEVANCE (See instructions): This project focuses on the reconstitution of donor CD4+ regulatory T cells (Treg) after allogeneic hematopoietic stem cell transplantation (HSCT). These cells are essential for establishing and maintaining immune tolerance and therefore play an important role in allo-immunity and tumor immunity. Clinical trials designed to modulate these cells in vivo are already in place and studies in this project will define the effects of these treatments on Treg in patients after HSCT.